Since many gene products such as RNA and proteins are enriched in regions where they function, their location provides an important clue to their function. This property has been used for in situ fluorescent hybridization, immunohistochemistry and tissue-specific reporter assays in numerous areas of biological research. Most optical sequencing methods either utilize a two-dimensional solid substrate or microwells/microchambers to immobilize sequencing templates during the sequencing process in order to maintain spatial invariability for optical detection, and therefore enable reconstruction of a single nucleic acid template sequence. In both cases, optical detection of signal occurs in at most two two-dimensional planes.